Inserm, U982, University of Rouen, Mont-Saint-Aignan, France; Laboratory of Neuronal and Neuroendocrine Differentiation and Communication, Institute for Research and Innovation in Biomedicine (IRIB), University of Rouen, Mont-Saint-Aignan, France; Normandy University, University of Rouen, Mont-Saint-Aignan, France.

Abstract

Urotensin II (UII) is an evolutionarily conserved neuropeptide initially isolated from teleost fish on the basis of its smooth muscle-contracting activity. Subsequent studies have demonstrated the occurrence of several UII-related peptides (URPs), such that the UII family is now known to include four paralogue genes called UII, URP, URP1 and URP2. These genes probably arose through the two rounds of whole genome duplication that occurred during early vertebrate evolution. URP has been identified both in tetrapods and teleosts. In contrast, URP1 and URP2 have only been observed in ray-finned and cartilaginous fishes, suggesting that both genes were lost in the tetrapod lineage. In the present study, the distribution of urp1 mRNA compared to urp2 mRNA is reported in the central nervous system of zebrafish. In the spinal cord, urp1 and urp2 mRNAs were mainly colocalized in the same cells. These cells were also shown to be GABAergic and express the gene encoding the polycystic kidney disease 2-like 1 (pkd2l1) channel, indicating that they likely correspond to cerebrospinal fluid-contacting neurons. In the hindbrain, urp1-expressing cells were found in the intermediate reticular formation and the glossopharyngeal-vagal motor nerve nuclei. We also showed that synthetic URP1 and URP2 were able to induce intracellular calcium mobilization in human UII receptor (hUT)-transfected CHO cells with similar potencies (pEC50=7.99 and 7.52, respectively) albeit at slightly lower potencies than human UII and mammalian URP (pEC50=9.44 and 8.61, respectively). The functional redundancy of URP1 and URP2 as well as the colocalization of their mRNAs in the spinal cord suggest the robustness of this peptidic system and its physiological importance in zebrafish.

urp1 mRNA occurs in cells located along the ventral edge of the central canal of spinal cord in adult zebrafish.

Expression of urp1 revealed by ISH (BM purple, violet) on free-floating sections of adult spinal cord. urp1+ cells form a quasi-continuous line at the ventral edge of the central canal (A). urp1+ cells are in close contact to the lumen of the central canal (arrowhead) (B). A1 and A2, lateral sections with dorsal up; B, coronal section with dorsal up. urp1+ cells boxed in A1 are shown in A2 at higher magnification. M, melanocytes. Scale bars: 50 μm.

urp1 and urp2 mRNAs are mainly coexpressed in the same spinal cord cells in zebrafish.

Simultaneous expression of urp1 and urp2 revealed by double fluorescent ISH (TAMRA, red for urp1, FITC, green for urp2 and DAPI in blue) on 24 hpf-embryo (A) and adult spinal cord sections (B, C). In embryo, all the stained cells contain both urp1 and urp2 mRNAs (A). In adult, although most of the stained cells are doubly-positive for urp1 and urp2 (arrow), some of the urp2+ cells are devoid of any urp1 mRNA (arrowhead). The white dash line indicates the central canal. A, dorsal view; B, sagittal section with dorsal up; C, coronal section with dorsal up. Scale bars: 15 μm.

Simultaneous expression of urp1 and pkd2l1 revealed by double fluorescent ISH (TAMRA, red for urp1 and FITC, green for pkd2l1) on 24 hpf-embryo (A) and adult spinal cord sections (B). pkd2l1 mRNA is distributed in two rows of cells along the rostro-caudal axis of the spinal cord both in embryo and adult (A2, B2). All the urp1+ cells are pkd2l1+(A1,3, B1,3) but only the ventral pkd2l1+ cells are urp1+. The white dash line indicates the central canal. A, lateral views; B, sagittal sections with dorsal up. Scale bars: 20μm.

urp1+ cells express isl1 and ss1, two markers of the vagus motor nucleus in zebrafish embryo.

Simultaneous expression of urp1 and isl1(A) or ss1(B) revealed by double fluorescent ISH (TAMRA, red for urp1 and FITC, green for isl1 or ss1) on 48 hpf-embryo. urp1+ cells are located at the level of the medial motor nucleus of the vagus. Most of them appear to be both isl1+ and ss1+. All pictures are dorsal views with anterior left. The boxed region in A1 is shown at higher magnification in A2–A4 and the same region is shown in B. nV, trigeminal nerve motor nuclei; nVII, facial nerve motor nuclei; nX, vagal nerve motor nuclei. Scale bars: 20 μm.

URP1 and URP2 are equipotent to induce intracellular calcium mobilization in a hUT-transfected CHO cell line.

Representative dose-response curves of hUII (●), mURP (■), URP1 (▲) and URP2 (▼) on the intracellular calcium mobilization (A). The values are expressed as percentages of the baseline and each point is the mean (± S.E.M.) of 3 replicates. Experimental data were fitted using a four-parameter logistic equation. The potencies of 7–13 independent experiments for each peptides were plotted as—Log(EC50) with box and whiskers (B). Values were considered as statistically different as assessed by analysis of variance followed by Tukey’s post-test, n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.